JP2016210795A - Methods for treating acute coronary syndrome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods and therapeutic agents for treating or managing acute coronary syndrome, particularly, unstable angina and acute myocardial infarction.SOLUTION: Provided is a therapeutic formulation for treating acute coronary syndrome comprising a therapeutic agent which specifically decreases or inhibits the activity of phagocytic cells and/or eliminates or diminishes the amount of phagocytic cells including but not limited to macrophages and monocytes, where the therapeutic agent is encapsulated, embedded or in a particulated form of 0.05-1.0 micron in size, and the therapeutic formulation is administered to a patient in need thereof in an effective amount to decrease the phagocytic activity. In some embodiments of the formulation, the therapeutic agent is a bisphosphonate represented by Formula (I). (Ris H, OH or halogen; Ris halogen, heteroaryl, cycloalkylamino etc.)SELECTED DRAWING: None

Description

1. This application is a continuation-in-part of US Application No. 10/607623 filed June 27, 2003, which is incorporated by reference in its entirety.

The present invention relates to methods and compositions designed for the treatment or management of acute coronary syndrome, particularly unstable angina and acute myocardial infarction. The methods of the invention include one or more treatments that specifically reduce or inhibit the activity of phagocytic cells, including but not limited to macrophages and monocytes, and / or eliminate or reduce the amount thereof. Administration of an effective amount of a formulation containing the drug.

2. BACKGROUND OF THE INVENTION Coronary artery disease is the leading cause of death in industrialized countries. In the United States, 50-60% of heart attacks occur in people who do not have a proven coronary artery disease. The main contributing factor to the pathology of this disease is the formation of atheroma plaques. Atherosclerotic plaque is a thickened part of the vessel wall due to the accumulation of cholesterol, proliferating smooth muscle cells and inflammatory cells.

Atherosclerotic plaque In general, atherosclerotic plaques consist of localized bulge sites with a central core of extracellular lipids covered by a fibrous cap. The core in the atheroma plaque contains crystalline cholesterol, cholesterol esters, phospholipids, cell degradation products and collagen remnants. Fibrous capsule separates the core of atherosclerotic plaque from the lumen of blood vessels or arteries and from connective tissue, which is a dense fibrous extracellular matrix composed of collagen, elastin, proteoglycans and other extracellular matrix materials It is mainly composed. Fibrous capsules differ in thickness, number of smooth muscle cells and macrophages, and collagen content (Vallabhajosula et al., 1997, J. Nucl. Med. 38 (11): 1788-1796).

Atherosclerotic plaques can be characterized as active and prone to rupture (“fragile or high risk plaques”) or inactive and relatively stable (“stable plaques”). Vulnerable, high-risk or ruptured plaques are characterized by numerous inflammatory cells (such as macrophages), a thin fibrous capsule and a large lipid core. The size of the lipid pool within the atheroma plaque and the thickness of the fibrous cap that covers it are important properties that predict plaque stability. The edge of the fibrous cap (shoulder portion) is a highly stressed location, and in part, the accumulation of inflammatory cells (such as macrophages) and degradation of the material that makes up the fibrous cap, which can lead to plaque rupture Secretion of the enzyme that causes (Moreno et al, Circulation, 1994, 90: 775-8; van der Wal et al., 1994, Circulation, 89: 36-44; Jander et al, 1998, Stroke, 29: 1625-1630 ), Easy to burst.

The rupture of lipid accumulating plaques exposes the highly thrombogenic core and arterial wall subendothelial vascular smooth muscle cell components to circulating blood. Platelet activation, adhesion and aggregation follows almost immediately. Platelet adhesion and activation results in the release of coagulation factors and the initiation of the coagulation cascade. Released growth factors, particularly platelet-derived growth factor (PDGF), stimulate the proliferation and migration of vascular smooth muscle cells. Vascular smooth muscle cell proliferation and migration can lead to increased plaque modeling and vascular stenosis, or can interact with platelets resulting in increased thrombus formation (Pasterkamp et al., 2000, J, Clin. Basic Cardiol. 3: 81-86). The resulting thrombosis caused by vulnerable plaque can lead to unstable angina, acute myocardial infarction, stroke, acute exacerbation of peripheral arterial disease or sudden coronary death.

An unstable angina heart requires oxygen-rich blood to function. The right and left coronary arteries branch off from the aorta and carry oxygenated blood to the heart tissue. When the coronary arteries cannot deliver a sufficient amount of oxygen-rich blood to the heart (a condition called hypoxia), chest pain, pressure or discomfort commonly known as angina occurs. If this condition persists, the myocardium itself can be damaged reversibly or irreversibly due to worsening oxygen conditions (a condition known as ischemia).

Angina is broadly classified as stable or unstable depending on the severity and pattern of occurrence. Stable angina occurs when increased physical activity (eg, rushing the road or going up a long staircase) increases the demand for oxygen-rich blood. Due to the multiple factors that may be present, the most common being one or more occluded coronary arteries, the supply provided by coronary blood flow cannot meet the increased demand and hypoxia occurs. Unstable angina is understood as angina pain that occurs with a lower degree of physical movement, more frequently, or at rest (ie, without physical movement). Unstable angina that occurs at rest is its most serious form of condition. This is usually caused by the formation of blood clots in the coronary arteries at the site of unruptured plaque, which can lead to heart attacks and irreversible damage to the heart if left untreated.

Unstable angina can result from partial rupture of vulnerable plaque that has become unstable. Partial rupture of the plaque generates a thrombus but does not completely occlude the artery. Although the endogenous clot-fitting mechanism serves to break up the clot, over time, the plaque continues to rupture and the clotting episode repeats. The patient may not have had myocardial infarction yet, but the risk is high (eg, if the vulnerable plaque ruptures completely, or if the intrinsic clot struggle mechanism is prior to complete occlusion of the artery If you can't remove it). Disrupted fibrous capsules collected post-mortem from patients with unstable angina are often more heavily infiltrated by macrophages at plaque rupture sites than plaques in stable angina cases.

Acute myocardial infarction Acute myocardial infarction (“AMI”) refers to a common clinical condition that leads to necrosis of myocardial tissue. This condition is well known in the art and includes the occurrence of pain (in most cases intrathoracic), characteristic electrocardiographic changes and intracellular enzymes released by necrotic heart tissue (creatinine phosphokinase and α- (Such as hydroxybutyric acid) or cardiac proteins (such as components of the troponin complex and myoglobin) are characterized by increased plasma concentrations. AMI may be accompanied by hypotension, circulatory failure, pulmonary edema and arrhythmia. In most, if not all, cases, AMI results from vascular damage and thrombosis in the coronary vessels that cause the blood vessels to become occluded and then cause impaired blood flow to the compromised myocardium (Fuster et al., 1992, New Engl. J. Med., 326: 242-310). In most cases, the time of occlusion of the coronary vessels can be estimated from the history, the course of plasma concentrations of myocardial enzymes and electrocardiographic changes.

The first event of many myocardial infarctions (heart attacks) is the rupture of atheroma plaques. Such a rupture can result in the formation of a thrombus or clot in the coronary artery supplying blood to the infarcted area. An infarcted area or region is a region of necrosis due to closed blood circulation, as is commonly said. The formed thrombus consists of a combination of fibrin and blood cells. The location, extent and duration of the occlusion caused by the clot determines the size of the infarct region and the extent of injury. Ultimately, the degree of myocardial damage caused by coronary occlusion is the “region” that is blood-fed by the affected blood vessel, the degree of occlusion of the blood vessel, and the amount of blood supplied to the affected tissue by the collateral vessel and on the oxygen demand of myocardium blood supply is suddenly limited (Pasternak and Braunwald, 1994, Acute myocardial Infarction, Harrison's Principles of Internal Medicine, 13 th Ed., pgs.1066-77).

Macrophages and inflammatory responses Macrophages are involved in the cause and / or pathology of several coronary syndromes. Proteolytic protein macrophage secretion, which degrades the fibrous capsule of the plaque, reduces the thickness of the capsule and increases further macrophage infiltration, thereby contributing to plaque instability. Thus, macrophages are thought to have a central role in plaque rupture, and their high concentration is considered predictive of such rupture. Indeed, erosion and / or rupture of the atherosclerotic plaque capsule is known to alter arterial thrombus formation, leading to the occurrence of acute ischemic events. Clearly, rupture at the site of vulnerable atheroma plaques is the most frequent cause of acute coronary syndromes such as unstable angina, myocardial infarction or sudden death.

Inflammation has been linked to the pathogenesis of acute myocardial infarction and post-AMI healing and repair. Myocardial ischemia promotes an inflammatory response. In addition, reperfusion, which is the main current acute therapy for AMI, also promotes inflammation. Reperfusion involves the rapid dissolution of an occlusive thrombus and the return of blood flow to the heart region where its blood supply has been interrupted. The presence of inflammatory cells in ischemic myocardial tissue has traditionally been believed to be a pathophysiological response to injury. However, experimental studies have shown that the influx of inflammatory cells, specifically macrophages, which are phagocytic cells, into tissues leads to tissue damage beyond that caused only by ischemia, although essential for healing. It was.

Macrophages and other leukocytes infiltrate the myocardium immediately after ischemia. Macrophages secrete several cytokines that stimulate fibroblast proliferation. However, activated macrophages also secrete cytokines and other mediators that promote myocardial damage. Thus, influx of macrophages into the myocardium increases myocardial necrosis and enlarges the infarct area. Thus, although the acute phase of inflammation is a necessary response in the healing process, sustained activation is actually detrimental to the infarct region and surrounding regions, ie the so-called “peri-infarct zone”.

The inflammatory response after myocardial ischemia is very important in determining the severity of the resulting damage caused by activated macrophages. Plasma concentrations of inflammatory chemotactic factors (macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α)) were shown to correlate with subsequent heart failure and left ventricular failure. (See, eg, Parissis, et al., J. Interferon Cytokine Res., 22 (2): 223-9). Peripheral monocytosis (increased monocyte count) on days 2 and 3 after AMI is associated with left ventricular failure and left ventricular aneurysm, suggesting a role for monocytes in the development of left ventricular remodeling after reperfusion AMI (Maekawa Y. et al., 2002, J. Am. Coll. Cardiol., 39 (2): 241-6). Left ventricular remodeling after acute myocardial infarction is a process of infarct enlargement followed by progressive left ventricular dilatation with an adverse clinical outcome. Furthermore, plasma concentrations of macrophage chemoattractant protein-1 (MCP-1) are high in patients with acute myocardial infarction. MCP-1 is induced by myocardial ischemia / reperfusion injury and neutralization of this chemokine significantly reduces infarct size.

Inhibition of inflammatory responses by nonspecific anti-inflammatory complexes after coronary occlusion in several coronary occlusion / reperfusion models has been shown to reduce infarct extent (eg, Squadrito, et al., 1997, Eur J. Pharmacol, 335: 185-92; Libby, et al., 1973, J. Clin. Invest., 3: 599-607; Spath, et al., 1974, Circ. Res., 35: 44-51 See). However, these nonspecific therapies are associated with side effects such as scarring and healing inhibition and the occurrence of aneurysms and ventricular wall rupture in some patients. These therapies are therefore excluded from clinical use. However, animal models with low ability to suppress macrophage function due to deficiency of the anti-inflammatory cytokine interleukin-10 have been shown to suffer from increased infarct size and myocardial necrosis in the coronary occlusion model (Yang Z. et al., 2000, Circulation, 101: 1019-1026).
US Application No. 10/607623 Vallabhajosula et al., 1997, J. Nucl. Med. 38 (11): 1788-1796 Moreno et al, Circulation, 1994, 90: 775-8 van der Wal et al., 1994, Circulation, 89: 36-44 Jander et al, 1998, Stroke, 29: 1625-1630 Pasterkamp et al., 2000, J, Clin. Basic Cardiol. 3: 81-86 Fuster et al., 1992, New Engl. J. Med., 326: 242-310 Pasternak and Braunwald, 1994, Acute Myocardial Infarction, Harrison's Principles of Internal Medicine, 13thEd., Pgs. 1066-77 Parissis, et al., J. Interferon Cytokine Res., 22 (2): 223-9 Maekawa Y. et al., 2002, J. Am. Coll. Cardiol., 39 (2): 241-6 Squadrito, et al., 1997, Eur. J. Pharmacol, 335: 185-92 Libby, et al., 1973, J. Clin. Invest., 3: 599-607 Spath, et al., 1974, Circ. Res., 35: 44-51 Yang Z, et al., 2000, Circulation, 101: 1019-1026

One object of the present invention is to accumulate and / or from phagocytes (especially macrophages and monocytes) in patients suffering from acute coronary syndromes (especially unstable angina or / and myocardial infarction). Identification of therapeutic agents that can inhibit biological functions, including secretion of factors.

Another object of the present invention is the development of methods for treating acute coronary syndromes (particularly unstable angina or / and myocardial infarction) and stabilizing plaques associated with these syndromes.

3. SUMMARY OF THE INVENTION The present invention relates to methods and compositions designed for the treatment or management of acute coronary syndromes, particularly unstable angina and acute myocardial infarction. The methods of the present invention are effective for formulations comprising one or more therapeutic agents that specifically inhibit and / or reduce the activity of phagocytes, including but not limited to macrophages and monocytes. Including administration of an amount. Administration of a formulation comprising one or more therapeutic agents according to the present invention serves as an acute therapy aimed at stabilizing the patient's coronary syndrome. In one embodiment, a formulation comprising one or more therapeutic agents is administered to a patient suffering from unstable angina to stabilize vulnerable or unstable plaques. In other embodiments, the formulation is administered to patients currently suffering from or recently affected by acute myocardial infarction to minimize infarct size and myocardial necrosis.

In a preferred embodiment, the formulation specifically targets phagocytic cells. Because phagocytic cells have the unique ability of phagocytosis, in these embodiments, the formulation is prepared to include particles that are characterized to enter the cell primarily or exclusively by phagocytosis. The formulation may include an encapsulated therapeutic agent, an embedded therapeutic agent or a particulate therapeutic agent. Once phagocytosed, the therapeutic agent is released from the formulation into target phagocytic cells, such as macrophages and monocytes, inhibiting and / or destroying phagocytic function.

In one embodiment, the present invention relates to a method of treating acute coronary syndromes by administering to an individual in need thereof an effective amount of a formulation comprising an encapsulated therapeutic agent. The therapeutic agent is encapsulated in a suitable carrier of a specific size. The formulation specifically targets phagocytic cells by its properties such as size or charge, allowing the formulation to be taken up primarily or exclusively by phagocytosis. If the formulation is taken up by phagocytes, the encapsulated therapeutic agent is released and the agent can inhibit and / or destroy the activity of the phagocytic cells.

In another embodiment, the invention relates to a method of treating acute coronary syndromes by administering to an individual in need thereof an effective amount of a formulation comprising an embedded therapeutic agent. The therapeutic agent is embedded in a suitable carrier of a specific size. The formulation specifically targets phagocytic cells by its properties such as size and / or charge, allowing the formulation to be taken up primarily or exclusively by phagocytosis. Once inside the phagocytic cell, the implanted therapeutic agent is released and the agent can inhibit and / or destroy the activity of the phagocytic cell.

In another embodiment, the invention relates to a method of treating acute coronary syndromes by administering to an individual in need thereof an effective amount of a formulation comprising a particulate therapeutic agent. The therapeutic agent is formed into microparticles of a specific size. The formulation specifically targets phagocytic cells by particulate properties such as size and / or charge, allowing the formulation to be taken up primarily or exclusively by phagocytosis. Once inside the phagocytic cell, the particulate therapeutic can inhibit and / or destroy phagocytic activity.

The invention also relates to a method of stabilizing plaque associated with acute coronary syndromes by administering to an individual in need thereof an effective amount of a formulation comprising an encapsulated, embedded or particulate therapeutic agent.

In a further embodiment, the present invention provides a formulation selected from the group consisting of an encapsulated therapeutic agent, an embedded therapeutic agent and a particulate therapeutic agent for the treatment of acute coronary syndromes and a pharmaceutically acceptable excipient, carrier, stabilization A pharmaceutical composition for administration to a subject currently suffering from or recently suffering from acute coronary syndromes such as unstable angina and acute myocardial infarction, comprising an agent or diluent.

The formulations of the present invention are preferably 0.03 to 1.0 microns in size. However, depending on the type of drug and / or carrier used, more preferred ranges include, but are not limited to, 0.07-0.5 microns, 0.1-0.3 microns, and 0.1-0.18 microns. Not.

5. Detailed Description of the Invention Phagocytic cells, in particular macrophages and monocytes, are involved in the cause and / or pathology of several coronary syndromes. Macrophages / monocytes not only reduce the thickness of the capsule, but also degrade the plaque's fibrous capsule by the secretion of various substances that also serve to recruit additional macrophages / monocytes to the site. Degradation of the fibrous cap leads to exposure of the plaque's lipid core to blood as well as the initiation of a coagulation cascade that results in a thrombus. A thrombus may partially occlude the lumen and cause unstable angina, or may completely occlude the lumen and cause acute myocardial infarction. If an acute myocardial infarction occurs, macrophages / monocytes are recruited to damaged myocardial tissue and secrete cytokines and other mediators that promote myocardial damage and thereby cause more tissue damage than is caused by ischemia alone And increase myocardial necrosis to enlarge the infarct area. Although complete and chronic inactivation and / or removal of phagocytic cells is undesirable, such a decrease in phagocytic activity and / or presence is intended to stabilize the patient and / or reduce coronary syndrome disorders. Desirable for short periods during or after acute coronary syndrome.

The present invention relates to acute phagocytic cells during or after acute coronary syndrome for the treatment or management of acute coronary syndromes, including but not limited to unstable angina and acute myocardial infarction. It relates to methods and compositions designed to reduce or inhibit the activity of and / or eliminate or reduce the activity of macrophages and monocytes, including but not limited to. The methods of the present invention include one or more that specifically reduce or inhibit the activity of phagocytic cells (including but not limited to macrophages and monocytes) and / or eliminate or reduce the amount thereof. Administration of an effective amount of the formulation containing the therapeutic agent. Administration of a formulation comprising one or more therapeutic agents is considered acute short-term therapy aimed at patient stabilization and / or minimization of immediate and long-term disability due to acute coronary syndromes. In one embodiment, a formulation comprising one or more therapeutic agents to stabilize vulnerable or unstable plaque in patients suffering from unstable angina and reduce the immediate threat of acute myocardial infarction Administer. In other embodiments, one or more therapeutic agents are administered to patients currently suffering from or recently affected by acute myocardial infarction to minimize infarct size and myocardial necrosis.

The preparation used in the method of the present invention specifically reduces or inhibits the activity of phagocytes in a patient and / or eliminates or reduces the amount thereof. The specificity of the formulation is due to the ability of the composition to act only on specific cell types (eg, macrophages and / or monocytes). In a preferred embodiment, the specificity of the formulation for phagocytic cells is due to the physicochemical properties of the formulation, such as size or charge, which can be exclusively or primarily subject to phagocytic internalization. Once undergoing phagocytosis and entering the cell, the therapeutic agent inhibits or reduces phagocytic activity and / or destroys the phagocytic cell. While not intending to be bound by a specific mechanism of action, the therapeutic agent of the formulation is released when it enters the cell before it becomes dysfunctional and / or destroyed. .

Formulations of the invention, such as encapsulated therapeutic agents, embedded therapeutic agents or particulate therapeutic agents, temporarily deplete and / or inactivate cells, i.e. macrophages and / or monocytes, which are important triggers in the inflammatory response. Suppresses the inflammatory response. Encapsulated drugs, embedded drugs or particulate drugs are taken up by macrophages and monocytes by phagocytosis. In contrast, non-phagocytic cells cannot take up the formulation due to the large size of the formulation and / or other physicochemical properties.

As used herein, the term “phagocytosis” refers to a preferred means of entry into phagocytic cells and is well understood in the art. However, the term should be understood to include other forms of endocytosis that also achieve the same effect. In particular, it is understood that phagocytosis, receptor-mediated endocytosis and other cellular means of absorbing / internalizing substances from outside the cell are also encompassed by the methods and compositions of the present invention.

The present invention relates to a pharmaceutical composition comprising one or more therapeutic agents of the present invention for administration to a subject currently suffering from or recently suffering from acute coronary syndromes such as unstable angina and acute myocardial infarction. Also provide.

5.1 Therapeutic Agents The therapeutic agents used in the formulations and methods of the present invention specifically reduce or inhibit phagocytic activity in the patient and / or the phagocytic activity in the patient, depending on the size or charge of the formulation. Loss or reduce the amount of cells. A therapeutic that inhibits, destroys, suppresses, modifies and / or alters phagocytic cells so that once they enter phagocytic cells such as macrophages or monocytes, they no longer function normally and / or survive. It may be a substance, an inactivating substance, a toxin, an inhibitory substance and / or a cytostatic / cytotoxic substance.

As used herein, the term “therapeutic agent” refers to a formulation or part of a formulation that renders the formulation inactive / toxic, eg, inhibits or reduces phagocyte activity. And / or a molecule that removes or reduces the amount of phagocytic cells. Compounds that can be therapeutic agents include inorganic or organic compounds; small molecules (less than 500 daltons) or large molecules including but not limited to inorganic or organic compounds; peptides, polypeptides, proteins, post-translationally modified proteins, antibodies, etc. Proteinaceous molecules including, but not limited to: nucleic acid molecules including but not limited to double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA or triple helix nucleic acid molecules, It is not limited to. The compound may be a natural product obtained from any known organism (including but not limited to animals, plants, bacteria, fungi, protists or viruses) or from a library of synthetic molecules. The therapeutic agent may be a monomer or a polymerized compound.

In preferred embodiments, the preferred therapeutic agent may be a bisphosphonate or an analog thereof. As used herein, the term “bisphosphonate” refers to geminal and non-geminal bisphosphonates. In a specific embodiment, the bisphosphonate has the following formula (I):

Wherein R ₁ is H, OH or a halogen atom, and R ₂ is a linear or branched C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl optionally substituted with halogen, heteroaryl, or amino there primary, secondary or tertiary, which may be heterocyclyl _C 1 _{-C 10} alkylamino or _C 3 -C ₈ cycloalkylamino, Y is _hydrogen, C 3 -C ₈ cycloalkyl, aryl or heteroaryl —NHY, or R ₂ is —SZ, wherein Z is chloro-substituted phenyl or pyridinyl.

In a more specific embodiment, the bisphosphonate is alendronate or an analog thereof. In such embodiments, the alendronate has the following formula (II):

In other specific embodiments, additional bisphosphonates can be used in the methods of the invention. Examples of other bisphosphonates are clodronate, tiludronate, 3- (N, N-dimethylamino) -1-hydroxypropane-1,1-diphosphonic acid, such as dimethyl-APD, 1-hydroxy-ethylidene-1,1- Bisphosphonic acids such as etidronate, 1-hydroxy-3 (methylpentylamino) -propylidene-biphosphonic acid (ibandronic acid) such as ibandronate, 6-amino-1-hydroxyhexane-1,1-diphosphonic acid such as Amino-hexyl-BP, 3- (N-methyl-N-pentylamino) -1-hydroxypropane-1,1-diphosphonic acid, such as methyl-pentyl-APD, 1-hydroxy-2- (imidazol-1 -Yl) ethane-1,1-diphosphonic acid, for example, zolendronic acid, 1 Hydroxy-2- (3-pyridyl) ethane-1,1-diphosphonic acid (risedronic acid), for example risedronate, 3- [N- (2-phenylthioethyl) -N-methylamino] -1-hydroxypropane- 1,1-bisphosphonic acid, 1-hydroxy-3- (pyrrolidin-1-yl) propane-1,1-bisphosphonic acid, 1- (N-phenylaminothiocarbonyl) methane-1,1-diphosphonic acid, for example FR78844 (Fujisawa), 5-benzoyl-3,4-dihydro-2H-pyrazole-3,3-diphosphonic acid tetraethyl ester, such as U81581 (Upjohn) and 1-hydroxy-2- (imidazo [1,2-a ] Pyridin-3-yl) ethane-1,1-diphosphonic acid, such as YM529 or an analogue thereof. But it is not limited to, et al.

Other formulations containing therapeutic agents include, but are not limited to, gallium, gold, selenium, gadolinium, silica, mitramycin, paclitaxel, sirolimus, everolimus and other similar analogs. In general, chemotherapeutic drugs such as 5-fluorouracil, cisplatin, alkylating agents, for example, other anti-proliferative or anti-inflammatory compounds such as steroids, aspirin and non-steroidal anti-inflammatory drugs can also be used in the formulation.

The present invention is meant to include the administration of one or more formulations for managing or treating acute coronary syndromes. Multiple formulations can be administered to a patient in combination. The term “in combination” is not limited to the exact simultaneous administration of the formulation, but allows the formulation to work together on the patient in turn so as to obtain higher efficacy when administered by other methods. Means administration within a reasonable time. For example, each formulation can be administered simultaneously or sequentially in a certain order at different times. However, if not administered at the same time, it should be administered in a time sufficiently close to obtain the desired therapeutic effect. Each formulation can be administered separately, in an appropriate manner, by an appropriate route that efficiently transports the therapeutic agent to the appropriate or desired site of action. Preferred methods of administration include intravenous (IV) and intraarterial (IA). Other suitable administration methods include intramuscular (IM), subcutaneous (SC) and intraperitoneal (IP) and oral (PO). Such administration may be a bolus injection or infusion. Another method of administration may be perivascular delivery. The formulation can be administered directly or after dilution. Combinations of any of the above routes of administration can also be used according to the present invention.

In various embodiments, the formulation is administered at intervals of less than 1 hour, at intervals of about 1 hour, at intervals of about 1 hour to about 2 hours, at intervals of about 2 hours to about 3 hours, at about 3 hours to about 4 hours. At intervals of about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about At intervals of 8 hours to about 9 hours, at intervals of about 9 hours to about 10 hours, at intervals of about 10 hours to about 11 hours, at intervals of about 11 hours to about 12 hours, at intervals of 24 hours or less, or 48 Dosing at sub-hour intervals. In one embodiment, two or more formulations are administered simultaneously or within the same patient visit.

5.1.1 Identification of therapeutic agents The present invention provides methods of screening for compounds that can be used as therapeutic agents. While not intending to be bound by a particular mechanism of action, compounds that are therapeutic agents for use in the methods of the present invention may be targeted by phagocytic cells due to the physicochemical properties of the formulation itself. I) inhibit phagocytic activity, ii) reduce phagocytic activity, iii) remove phagocytic cells from the circulation and / or from areas affected by acute coronary syndrome, and / or iv) in circulation and The number of phagocytic cells in an area affected by acute coronary syndrome can be reduced.

Methods for screening for therapeutic agents generally involve incubating a candidate compound with a phagocytic cell in vitro or in vivo, and then measuring a change (eg, decrease) in phagocytic activity or life span, thereby resulting in the present invention. Identifying compounds that are therapeutic agents for use in. Any method known in the art can be used to measure phagocyte activity or life span. In one embodiment, phagocytic activity is measured by the level of cell activation relative to an activation stimulus. For example, macrophage / monocyte activation is induced by chemotactic factors such as macrophage chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) and interleukin 1β (IL-1β) and tissues It can be measured by quantifying the level of other substances produced by macrophages such as necrosis factor α (TNF-α). In other embodiments, phagocytic cell life is measured. For example, cell proliferation can be detected by measuring ³ H-thymidine incorporation and by direct cell counting to detect transcriptional activity of known genes such as proto-oncogenes (eg, fos, myc) or changes in cell cycle markers. Or by trypan blue staining. Measure mRNA transcription levels (eg, by Northern blot, RT-PCR, Q-PCR, etc.) or protein levels (eg, ELISA, Western blot, etc.) using any method known in the art. be able to.

In one embodiment, the compound that decreases phagocyte activity is:
a) contacting a phagocytic cell with a first compound that is a compound that activates the phagocytic cell and a second compound that is a candidate compound;
b) of activation in phagocytic cells (ie, control cells) identified by measuring the level of activation in the contacted phagocytes and contacted with the first compound in the absence of the second compound A decrease in activation in the contacted cells compared to the level indicates that the second compound decreases phagocytic activity.

In other embodiments, the compound that reduces the amount of phagocytes is
a) contacting phagocytes with the compound;
b) a decrease in the viability of the contacted cells as compared to the viability of the phagocytic cells that were identified by measuring the viability of the contacted phagocytes and not contacted with the compound (ie, control cells) , Indicating that the compound reduces the amount of phagocytic cells.

In other embodiments, the ability of a candidate compound to alter phagocyte activity or lifespan substantially similar or better than compounds known to alter phagocyte activity or lifespan in a therapeutically desirable manner. Measure. As used herein, “substantially similar to” refers to agents that have similar effects on phagocytes as exemplary agents, ie, agents that inhibit phagocyte activity, function, motility and / or depletion. .

In addition, candidate compounds can be used in animal models of acute coronary syndromes to assess their ability to be used in the methods of the invention. In one embodiment, a rabbit AMI model can be used (see, eg, section 6.1).

5.2 Formulation of a therapeutic agent A formulation comprising one or more therapeutic agents is prepared to be large enough to be internalized by phagocytosis exclusively or primarily, thereby Specificity to cells can be conferred. If such a formulation enters the cell, non-phagocytic cells may be affected by such formulation, but there is no mechanism for non-phagocytic cells to internalize the formulation thus prepared. The size range of the formulations that confer exogenous specificity to one or more therapeutic agents is preferably 0.03-1.0 microns, more preferably 0.07-0.5 microns, more preferably 0.1 to 0.3 microns, more preferably 0.1 to 0.18 microns.

Any method known in the art can be used to incorporate a therapeutic agent into a formulation to undergo internalization exclusively or primarily by phagocytosis. The therapeutic agent formulation may sequester the therapeutic agent for a time sufficient to facilitate delivery of the agent to the target site. In addition, the therapeutic agent formulation can release the therapeutic agent from the particles when present in target cells (eg, phagocytic cells) at the target site.

In one embodiment, the therapeutic agent is encapsulated in a carrier with the desired properties (ie, an encapsulant). In certain embodiments, the encapsulating agent is a liposome. Liposomes can be prepared by any method known in the art (eg, Monkkonen J. et al., 1994, J. Drug Target, 2: 209-308; Monkkonen J. et al. , 1993, Calcif.Tissue Int., 53: 139-145; Lasic DD., Liposomes Technology Inc., Elsevier, 1993, 63-105. (Chapter 3); Winterhalter M, Lasic DD, Chem Phys Lipids, 1993 Sep; 64 (1-3): 35-43). Liposomes may be positively charged, neutral or more preferably negatively charged. Liposomes can be lipid monolayers or multi-layered. Suitable liposomes according to the present invention are preferably non-toxic liposomes such as those prepared from phosphatidylcholine phosphoglycerol and cholesterol. The diameter of the liposome used is 0.03 to 1.0 μm. However, other size ranges suitable for phagocytosis by phagocytic cells can also be used.

In other embodiments, the therapeutic agent is embedded in a carrier of desired properties (ie, an embedding agent). The therapeutic agent to be embedded is a therapeutic agent that is embedded, encapsulated and / or absorbed in a carrier, dispersed in a carrier matrix, adsorbed and / or bound on a carrier surface, or any combination of these forms. Etc. In certain embodiments, the embedding agent (or carrier) is a microparticle, nanoparticle, menosphere, microsphere, microcapsule or nanocapsule (eg, M. Donbrow in: Microencapsulation and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, FL, 347, 1991). The term carrier includes polymeric and non-polymeric preparations. In certain embodiments, the embedding agent is a nanoparticle. Preferably, the nanoparticles are 0.03 to 1.0 microns in diameter and may be spherical, non-spherical or polymeric particles. The therapeutic agent may be embedded in the nanoparticles, uniformly or non-uniformly dispersed in the polymer matrix, adsorbed on the surface, or any combination of these forms. In a preferred embodiment, the polymer used to produce the nanoparticles is biocompatible and biodegradable, such as poly (DL-lactide coglycolide) polymer (PLGA). However, other polymers that can be used to produce nanoparticles include PLA (polylactic acid) and their copolymers, polyanhydrides, polyalkyl cyanoacrylates (polyisobutyl cyanoacrylates), polyethylene glycols, Including but not limited to polyethylene oxide and derivatives thereof, chitosan, albumin, gelatin and the like.

In other embodiments, the therapeutic agent is particulate and each of the particles has the desired properties. Particulate therapeutic forms include those in the form of insoluble suspended or dispersed microparticles that are not encapsulated, embedded or absorbed in a carrier. Particulate therapeutic agents include therapeutic agents that are drug suspension or dispersion colloids, aggregates, flocculent aggregates, insoluble salts, insoluble complexes and polymeric chains. Such microparticles are insoluble in the fluid in which they are stored / administered (eg, saline or water) as well as the fluid in which they exhibit their therapeutic effect (eg, blood or serum). In general, “insoluble” refers to the solubility of 1 part particulate therapeutic in more than 10,000 parts of solvent. Methods known in the art for preparing microparticles or aggregates can be used. Preferably, the microparticles are 0.03-1.0 microns in diameter and may be in any particulate form.

5.2.1 Determination of particle size Formulations containing therapeutic agents are prepared such that the size of the formulation is large enough to undergo internalization exclusively or primarily by phagocytosis, ie greater than 0.03 microns. It is preferable to do. In preferred embodiments, such formulations are preferably 0.03-1.0 microns, more preferably 0.07-0.5 microns, more preferably 0.1-0.3 microns, most preferably 0. .1 to 0.18 microns. Prior to administration to a patient in need thereof, the size of the formulation can be measured using any method known in the art. For example, a Nicomp Submicron Particle Sizer (model 370, Nicomp, Santa Barbara, CA) using laser light scattering can be used.

5.3 Administration of the formulation An effective amount of the formulation is considered as a short-term acute therapy and is not intended for chronic administration. The dosing period is such that it results in inhibition / depletion of phagocytes for less than 1 month, preferably less than 2 weeks, most preferably up to 1 week. Empirically, this can be determined by administering the compound to an individual in need thereof (or an animal model of such an individual) and monitoring the level of inhibition / depletion at various time points. The time of inhibition can also be correlated with an appropriate desired clinical effect, eg, a reduction in the acute risk of plaque rupture.

5.4 Characterization of therapeutic utility The term “effective amount” achieves the desired therapeutic result, ie inhibition or reduction of the activity of phagocytes and / or disappearance or reduction of the amount of phagocytes. Means the amount of a particular formulation effective above. In one embodiment, the desired therapeutic outcome of inhibiting or reducing the activity of phagocytes and / or eliminating or reducing the amount of phagocytes is fragile or unstable plaque in a patient suffering from unstable angina To stabilize. In other embodiments, the desired therapeutic outcome of inhibiting or reducing the activity of phagocytes and / or eliminating or reducing the amount of phagocytes is the infarct size and / or in patients suffering from acute myocardial infarction. Minimize the amount of myocardial necrosis.

Toxicity and efficacy of the treatment methods of the present invention include, for example, LD ₅₀ (a lethal dose relative to 50% of the population), maximum non-toxic dose (NOAEL) and ED ₅₀ (50% of the population are therapeutically effective). Can be determined by standard pharmacological methods in cell culture or laboratory animals to determine the amount. The dose ratio between toxic and therapeutic effects is the therapeutic index, and formulations with large therapeutic indices that can be expressed as the ratio LD ₅₀ / ED ₅₀ or NOAEL / ED ₅₀ are preferred. Formulations that exhibit toxic side effects can be used, but to minimize the possibility of injury to non-affected cells and thereby reduce side effects, the drug target of such formulations is targeted to the affected tissue site. Care should be taken to design the delivery system that defines.

Data obtained from cell culture assays and animal studies can be used to determine the dosage range of formulations used in humans. The dosage of such formulations is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For formulations used in the methods of the invention, the effective amount can be estimated initially from cell culture assays. Doses that achieve circulating plasma concentrations, including the IC ₅₀ determined in cell culture (ie, the concentration of test compound that achieves half-maximal suppression of symptoms), can be formulated in animal models. Such information can be used to more accurately determine useful doses in humans. The plasma concentration can be measured, for example, by high performance liquid chromatography.

The protocols and compositions of the invention are preferably tested in vitro and then in vivo for the desired therapeutic activity prior to use in humans. One example of such an in vitro assay is grown in culture, exposed, or otherwise administered to a cell and the effect of this assay on the cell, eg, inhibition or reduction of activity and / or complete or In vitro cell culture assay in phagocytic cells to observe for partial cell death. Phagocytic cells can be obtained from established cell lines or can be isolated from an individual recently as a primary cell line. Many standard assays in the art can be used to measure the activity of a formulation against phagocytes. For example, macrophage / monocyte activation is induced by macrophage chemoattractant protein-1 (MCP-1), interleukin 1β (IL-1β), tissue necrosis factor α (TNF-α) and macrophage inflammatory protein-1α (MIP− It can be measured by quantifying the level of chemotactic factors such as 1α). A number of standard assays in the art can be used to assess phagocyte survival and / or growth. For example, cell proliferation can be detected by measuring ³ H-thymidine incorporation and by direct cell counting to detect transcriptional activity of known genes such as proto-oncogenes (eg, fos, myc) or changes in cell cycle markers. The cell viability can be measured by trypan blue staining.

The selection of a preferred effective amount can be determined by one skilled in the art based on consideration of several factors known to those skilled in the art (eg, by clinical trials). Such factors include those known to those skilled in the art to reflect the acute coronary syndrome to be managed or treated, the accompanying symptoms, the patient's physique, the patient's immune status and the accuracy of the administered pharmaceutical composition. Factors are mentioned.

5.5 Pharmaceutical Compositions and Routes of Administration Formulations containing one or more therapeutic agents for use in the methods of the present invention may vary according to various factors specific to each patient (eg, severity and type of patient disorder, age, , Body weight, response and past medical history), number and type of therapeutic agents in the formulation, type of formulation (eg, encapsulation, embedding, particulate, etc.), form of the composition (eg, liquid, semi-liquid or solid) And / or route of administration (e.g., oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, Depending on the vagina or rectum, it may take many forms. Water, saline solution, buffered saline solution, oil (eg petroleum, animal, vegetable or synthetic oil), starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, white ink, silica gel, sodium stearate, mono Pharmaceutical compositions, excipients, additives or diluents, including but not limited to glycerol stearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, ethanol, dextrose, etc. are included in the compositions of the present invention be able to. If desired, the composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release agents and the like.

Formulations suitable for parenteral administration can be prepared as aqueous solutions, preferably using physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or excipients are fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or substances that increase the solubility of the compounds that allow the preparation of highly concentrated solutions.

The pharmaceutical composition can be provided as a salt and can be produced using a number of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base form.

The pharmaceutical composition can be administered systemically or locally, for example, near the site of an acute coronary syndrome condition. Further, systemic administration is meant to include administration that can be targeted to a specific area or tissue type of interest.

The pharmaceutical composition may be, for example, acute plaque such as chest pain associated with plaque rupture, pain radiated to the shoulders, arms, teeth, jaw, abdomen or back or shortness of breath or cough, lightheadedness, fainting, nausea, vomiting, sweating or anxiety. It is preferred to administer immediately upon the occurrence of the first symptom of rupture. Other symptoms will be apparent to those skilled in the art and physicians, and may be a signal for administering the pharmaceutical composition of the present invention. Alternatively and / or additionally, the pharmaceutical composition can be administered immediately after the onset of symptoms, eg, within minutes of the onset of symptoms. Alternatively and / or additionally, the pharmaceutical composition may be within 1 hour after the onset of symptoms, or at about 2 hours, or at about 3 or 4 hours, or at about 5 or 6 hours. The eye can be administered within 1-3 days.

In other therapies, the pharmaceutical composition is administered to patients at high risk for plaque rupture. For example, the compositions of the invention can be administered to a patient prior to a procedure that increases the risk of plaque rupture, such as, for example, angioplasty. It is preferred that the composition be administered up to 3 days prior to such a procedure. Also preferably, administration may be from 1 to 6 hours before the procedure or within 1 hour of the procedure or less than 1 hour before or within a few minutes of the procedure. Those skilled in the art will recognize, for example, various physiological factors specific to an individual patient, such as weight, medical history and genetic predisposition, and various factors that affect the expected risk of plaque rupture, such as the complexity of the procedure being performed. This makes it easy to determine the appropriate time of administration.

All published articles, books, reference manuals and abstracts cited herein are hereby incorporated by reference in their entirety to more fully describe the state of the art to which this invention pertains.

Since various modifications can be made to the above objects without departing from the scope and spirit of the invention, all objects contained in the above description or defined in the appended claims It is intended that this invention be interpreted and described. Modifications and variations of the present invention are possible in light of the above teachings.

6). EXAMPLES The following examples described herein are meant to illustrate and illustrate various aspects of practicing the invention and are intended to limit the invention in any way. It is not a thing.

6.1 Effect of Liposomal Alendronate on Infarct Area Size The effect of administration of encapsulated bisphosphate on infarct area size was tested in a rabbit AMI model. Liposomal alendronate with a diameter of about 0.150 μm was prepared using the following outline.
a. Lipids, DSPC, DSPG and cholesterol are dissolved in 1/1 ethanol / tert-butanol.
b. A buffer containing alendronate is diluted with a solvent to generate large multilamellar vesicles (MLVs).
c. MLVs are extruded through 200 nm polycarbonate filters to obtain large unilamellar 150 ± 20 nm vesicles (LUVs).
d. LUVs are ultrafiltered to remove unencapsulated alendronate.
e. Sterile filter.

Eight New Zealand White male rabbits weighing 2.5-3.5 kg were allowed free access to normal feed and water. Rabbits were randomly administered with saline (control) or liposomal alendronate (3 mg / kg, iv) as a single injection concomitant with coronary artery occlusion. Rabbits were anesthetized with ketamine / xylazine (35 mg / kg, 5 mg / kg) and isoflurane. Experiments were performed under intubation and respiratory support with mechanical ventilation using isoflurane in balanced oxygen and continuous echocardiography (BCG) and arterial blood pressure (intra-arterial catheter) monitoring. After performing chest surgery through the fourth intercostal space, pericardiotomy and pericardial cradle construction were performed. The left main coronary trunk was identified and the large branch was surrounded by 5-0 silk suture and snare. The snare was then tightened for 30 minutes. Ischemia was confirmed by ECG change (ST-T partial increase), segment color change and decreased motion. After 30 minutes, the snare was opened and resumption of blood flow was confirmed. The suture was left in place, loosened, and the chest cavity was closed layer by layer. Buprenex was administered to rabbits for an additional 2-3 days for analgesia. After anesthesia with Pental, the rabbits were sacrificed 7 days later and the hearts were collected. After perfusion of the coronary artery with saline through the ascending aorta, tighten the suture on the previously closed coronary artery and perfuse the coronary artery with 0.5% Evans blue solution (Sigma) to re-endothelialize The area was stained (presence of blood). The left ventricular area unstained by Evans Blue was defined as the area at risk. The heart was then frozen at -20 ° C for 24 hours and cut into transverse sections spaced 2 mm apart. Thin sections of the heart are incubated for 30 minutes in vital stain tritetrazolium chloride (TTC, 1%, Sigma) and fixed with 10% neutral buffered formalin to stain viable cells prior to tissue processing. did. The left ventricular region (white) that was not stained by TTC was defined as the infarcted region. The stained sections were then photographed and processed by digital planimetry (Photoshop).

Rabbits receiving liposomal alendronate had an infarct area of 29.5 ± 6% of the area at risk. This was in contrast to control rabbits (not receiving liposomal alendronate) that showed an area of infarct of 42 ± 5.5% of the area at risk (FIG. 1). Therefore, liposomal alendronate was effective in reducing the area of infarction. No side effects were observed in the administration group.

6.2 Effect of liposomal alendronate on myocardial morphology Rabbits treated in section 6.1 showed changes in myocardial morphology as shown by hematoxylin and eosin staining. Control rabbits have a distorted myocardial morphology (FIG. 2A), while rabbits administered liposomal alendronate show a more normal morphology (FIG. 2B).

6.3 Effect of Liposomal Alendronate on Macrophage Infiltration Rabbits treated in section 6.1 showed reduced macrophage infiltration in rabbits administered liposomal alendronate. Representative sections of rabbit hearts were subjected to immunostaining for RAM11 + macrophages. Rabbit sections receiving liposomal alendronate (FIG. 3B) showed less staining than control rabbit sections (FIG. 3A) and therefore had less RAM11 + macrophage accumulation.

Liposomal alendronate has also been shown to reduce the number of systemic circulating monocytes. Rabbits were administered saline (control) or liposomal alendronate (3 mg / kg, iv). Circulating monocyte levels were measured using FACS analysis of CD-14. 48 hours after the injection of liposomal alendronate, the blood monocyte population was reduced by 75-95% compared to the control group.

It is a figure which shows the effect of the liposomal arenate administration with respect to the magnitude | size of the infarction area | region after transient coronary artery occlusion in a rabbit. The size of the infarct region was calculated as the area of the infarct region as a percentage of the left ventricular area that is blood-fed by the occluded artery and is at risk for subsequent infarctions. Data are expressed as mean ± SD, n is 4 per group and p-value is p <0.05. FIG. 6 is a diagram of control rabbits showing the effect of liposomal alendronate administration on myocardial morphology after reversible coronary occlusion in rabbits. Control rabbits (A) have a distorted myocardial morphology, while rabbits (B) administered liposomal alendronate have a more normal myocardial morphology. FIG. 4 is a diagram of control rabbits showing a reduction in macrophage infiltration after administration of liposomal alendronate after reversible coronary occlusion in rabbits. The control rabbit (A) shows an increased accumulation of RAM11 + macrophages in the infarct area compared to the rabbit (B) administered liposomal alendronate.

Claims (12)

An effective amount of a formulation that reduces phagocytic activity, including a therapeutic agent that is encapsulated, embedded, or in particulate form with a size of 0.05 to 1.0 microns is administered to a patient in need thereof A therapeutic agent for acute coronary syndrome, comprising: An effective amount of a formulation that reduces the number of phagocytes, including a therapeutic agent that is encapsulated, embedded, or in particulate form with a size of 0.05 to 1.0 microns is administered to a patient in need thereof A therapeutic agent for acute coronary syndrome, comprising: The therapeutic agent according to claim 1 or 2, wherein the acute coronary syndrome is unstable angina. The therapeutic agent according to claim 1 or 2, wherein the acute coronary syndrome is imminent or impending or actual plaque rupture. The therapeutic agent according to claim 1 or 2, wherein the acute coronary syndrome is acute myocardial infarction. The therapeutic agent according to claim 1 or 2, wherein the therapeutic agent is a bisphosphonate. Bisphosphonate is represented by the following formula (I)


[Wherein R ₁ is H, OH or a halogen group,
R ₂ is a linear or branched C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl optionally substituted with halogen, heteroaryl, or amino is primary, secondary or tertiary heterocyclyl _C 1 _{-C 10} alkylamino or _C 3 -C ₈ cycloalkylamino, Y is _hydrogen, C 3 -C ₈ cycloalkyl, aryl or heteroaryl -NHY, or Z is chloro-substituted phenyl or pyridinyl -SZ]
The therapeutic agent of Claim 6 containing the compound which has this. The therapeutic agent according to claim 6, wherein the bisphosphonate is selected from the group consisting of clodronate, etidronate, tiludronate, pamidronate, alendronate, and risendronate. The therapeutic agent according to claim 1 or 2, wherein the therapeutic agent is encapsulated in liposomes. The therapeutic agent according to claim 1 or 2, wherein the therapeutic agent is embedded in a carrier selected from the group consisting of microparticles, nanoparticles, microspheres and nanospheres. The therapeutic agent according to claim 1 or 2, wherein the therapeutic agent is formulated as microparticles selected from the group consisting of aggregates, flocculent aggregates, colloids, polymer chains, insoluble salts and insoluble complexes. The therapeutic agent according to claim 1 or 2, wherein a plurality of therapeutic agents are contained in the preparation.